U.S. patent application number 12/621982 was filed with the patent office on 2010-05-20 for methods and processes for producing esters.
This patent application is currently assigned to THE OHIO STATE UNIVERSITY. Invention is credited to Shang-Tian Yang.
Application Number | 20100124773 12/621982 |
Document ID | / |
Family ID | 42172341 |
Filed Date | 2010-05-20 |
United States Patent
Application |
20100124773 |
Kind Code |
A1 |
Yang; Shang-Tian |
May 20, 2010 |
METHODS AND PROCESSES FOR PRODUCING ESTERS
Abstract
Methods are provided for producing esters. The methods comprise
converting a fermentable carbon source to organic acids by
fermentation with organic acid producing microorganisms, followed
by catalytic esterification. The methods comprise integrated
fermentation, extraction, and esterification reactions wherein the
organic acids produced during fermentation are extracted into an
extraction solvent and then directly reacted with an alcohol in the
presence of a catalyst to form organic esters. Methods of producing
esters are also provided wherein the organic acids produced during
fermentation and extracted into the extraction solvent are stripped
from the extraction solvent prior to being reacted with an alcohol
in the presence of a catalyst to form organic esters.
Inventors: |
Yang; Shang-Tian; (Dublin,
OH) |
Correspondence
Address: |
DINSMORE & SHOHL LLP
FIFTH THIRD CENTER, ONE SOUTH MAIN STREET, SUITE 1300
DAYTON
OH
45402-2023
US
|
Assignee: |
THE OHIO STATE UNIVERSITY
Columbus
OH
|
Family ID: |
42172341 |
Appl. No.: |
12/621982 |
Filed: |
November 19, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61116108 |
Nov 19, 2008 |
|
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|
Current U.S.
Class: |
435/135 |
Current CPC
Class: |
C12N 11/00 20130101;
C12N 9/1217 20130101; C12N 9/20 20130101; C12P 7/62 20130101; C12P
7/40 20130101 |
Class at
Publication: |
435/135 |
International
Class: |
C12P 7/62 20060101
C12P007/62 |
Claims
1. A method of producing esters comprising: fermenting a
fermentable carbon source in the presence of an organic acid
producing microorganism to produce fermentation output comprising
an organic acid; extracting the organic acid into an extractant;
and esterifying the organic acid in the extractant in the presence
of a catalyst and alcohol to produce an organic ester.
2. The method of claim 1, wherein the organic acid producing
microorganism comprises at least one of Clostridium tyrobutyricum,
Clostridium butyricum, Clostridium beijerinckii, Clostridium
populeti, Clostridium thermobutyricum, Rhizopus oryzae, and
Propionibacterium aidipropionici.
3. The method of claim 2, wherein the microorganism comprises
engineered mutants of Clostridium tyrobutyricum ATCC 25755 obtained
from inactivating the chromosomal ack gene encoding acetate
kinase.
4. The method of claim 1, wherein the step of fermenting the
fermentable carbon source in the presence of an organic acid
producing microorganism to produce fermentation output comprising
an organic acid is conducted at a pH from approximately 4 to 7.
5. The method of claim 1, wherein the fermentable carbon source is
derived from biomass feedstock.
6. The method of claim 5, wherein the biomass feedstock comprises a
carbohydrate source.
7. The method of claim 6, wherein the biomass feedstock comprises
at least one of agricultural residues and processing wastes.
8. The method of claim 7, wherein the agricultural residues
comprise corn stovers, corn cobs, and rice straw.
9. The method of claim 7, wherein the processing wastes comprise at
least one of cheese whey and corn fibers.
10. The method of claim 1, wherein the step of fermenting the
fermentable carbon source in the presence of an organic acid
producing microorganism comprises the use of a fibrous bed
bioreactor.
11. The method of claim 1, wherein the extractant is an amine
solvent.
12. The method of claim 11, wherein the amine solvent is a
long-chain aliphatic amine solvent.
13. The method of claim 1, wherein the catalyst is selected from
the group consisting of: sulfuric acid, a cation exchange resin,
and an enzyme.
14. The method of claim 13, wherein the enzyme is a lipase.
15. The method of claim 14, wherein the lipase is produced from
mutants of Candida lipolytica sp. 99-125.
16. The method of claim 14, wherein the lipase is immobilized on a
support surface.
17. The method of claim 16, wherein the lipase is immobilized on a
support surface comprising a fibrous matrix in a fibrous bed
bioreactor.
18. The method of claim 1, wherein the organic ester is stripped
from the extractant.
19. The method of claim 18, wherein the organic ester is stripped
from the extractant with steam in a distillation column.
20. A method of producing esters comprising: fermenting a
fermentable carbon source in the presence of an organic acid
producing microorganism to produce fermentation output comprising
an organic acid; extracting the organic acid into an organic
solvent; stripping the organic acid from the organic solvent; and
esterifying the organic acid in the presence of a catalyst and
alcohol to produce an organic ester.
21. The method of claim 20, wherein the step of stripping the
organic acid from the organic solvent comprises the use of a base
solution, a strong acid solution, hot water, or steam.
22. A method of producing esters comprising: fermenting a
carbohydrate source in the presence of engineered mutants of
Clostridium tyrobutyricum ATCC 25755 obtained from inactivating the
chromosomal ack gene encoding acetate kinase, to produce a
fermentation output comprising butyric acid, extracting the butyric
acid into an amine solvent; and esterifying the butyric acid in the
presence of alcohol and a lipase immobilized on a fibrous bed
bioreactor to produce a butyrate ester.
23. The method of claim 22, wherein the alcohol is at least one of
butanol or ethanol.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 61/116,108, filed Nov. 19, 2008, the contents
of which are hereby incorporated by reference in its entirety.
TECHNICAL FIELD
[0002] The present invention is generally directed to methods and
processes for producing organic acid esters, and more specifically
to methods of producing organic acid esters via fermentation,
extraction, and enzymatic esterification in an integrated process
to reduce process steps and production costs.
BACKGROUND
[0003] The conversion of organic acids and alcohols to
corresponding organic esters for industrial applications has been
widely studied. Organic esters are used in a wide variety of
applications, including the areas of biofuels, food flavors and
fragrances, and solvents.
[0004] Fermentation processes using microorganisms provide a
promising path for converting biomass and agricultural wastes into
chemicals and fuels. There are abundant low-value agricultural
commodities and food processing byproducts or wastes that require
proper disposal to avoid pollution problems. In the dairy industry,
approximately 80 billion pounds of cheese whey byproduct are
generated annually, much of which has no economical use and
requires costly disposal. Similarly, in the corn refinery industry,
more than 22% of the estimated 12 billion bushels (approximately
300 million metric tons) of corn annually produced in the United
States is processed to produce high-fructose-corn-syrup, dextrose,
starch, and fuel alcohol. It is thus desirable to convert these
byproducts and wastes to high-value products to reduce waste while
improving the process economics.
[0005] Bioethanol is the major biofuel currently available on the
market. Recently, however, biobutanol has attracted attention for
its potential as a transportation fuel because biobutanol is
noncorrosive and offers a safer fuel that can be dispersed through
existing pipelines and filling stations. As a biofuel, butanol has
the following advantages over ethanol: (a) butanol has 30% more Btu
per gallon; (b) butanol is less evaporative/explosive with a Reid
vapor pressure (RVP) 7.5 times lower than ethanol; (c) butanol is
safer than ethanol because of its higher flash point and lower
vapor pressure; and (d) butanol is more miscible with gasoline and
diesel fuel but less miscible with water.
[0006] Butyrate esters have similar energy content and properties
to biobutanol but offer the advantage of being easier to produce
than biobutanol. More specifically, butyrate esters have similar
energy content to butanol, are substantially insoluble in water,
and have lower vapor pressures and higher flash points than
ethanol. Butyrate esters may be produced from sugars via butyric
acid fermentation followed by esterification with an alcohol. Thus,
butyrate esters offer a novel alternative to existing biofuels.
[0007] Short-chain organic acid esters are also widely used as
flavor and fragrance compounds in food, beverage, cosmetic, and
pharmaceutical industries. Currently, most of the flavor compounds
are provided by traditional methods such as chemical synthesis or
extraction from natural sources. As a result, additional
embodiments for methods and processes for producing esters are
desired.
SUMMARY
[0008] The present invention relates generally to methods for
producing esters. According to one embodiment of the present
invention, the methods comprise converting a fermentable carbon
source to organic acids by fermentation with organic acid producing
microorganisms, followed by catalytic esterification. In a further
embodiment of the present invention, the methods comprise
integrated fermentation, extraction, and esterification reactions
wherein the organic acids produced during fermentation are
extracted into an extraction solvent and then directly reacted with
an alcohol in the presence of a catalyst to form organic esters.
The organic esters are then stripped from the extraction solvent
with steam in a distillation column. The extraction solvent is thus
regenerated and recycled for the extraction process, while the
ester and unreacted alcohol will be separated in the distillation
process.
[0009] In an alternative embodiment of the present invention,
methods for producing esters are provided wherein the organic acids
produced during fermentation and extracted into an extraction
solvent are stripped from the extraction solvent prior to being
reacted with an alcohol in the presence of a catalyst to form
organic esters.
[0010] These and other features and advantages of these and other
various embodiments according to the present invention will become
more apparent in view of the drawings, detailed description, and
claims provided that follow hereafter.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] The following detailed description of the embodiments of the
present invention can be best understood when read in conjunction
with the following drawings, where like structure is indicated with
like reference numerals, and in which:
[0012] FIG. 1 is a flow chart illustrating one embodiment of the
integrated fermentation-extraction-esterification process for the
production of butyrate esters.
[0013] FIG. 2 illustrates the construction of a fibrous bed
bioreactor (left) with enzyme immobilized on cotton fibers
(center), and the thermal stability of immobilized lipase compared
with free lipase at 60.degree. C. (right).
[0014] FIG. 3 is two graphs illustrating the kinetics of
esterification of butyric acid with ethanol (top) or butanol
(bottom) by immobilized lipase in a fibrous bed bioreactor.
[0015] FIG. 4 is a photograph of 2 flasks containing the colorless
ethyl butyrate (left) and butyl butyrate (right) esters produced
from esterification and distillation.
[0016] FIG. 5 shows the reaction mechanism of the esterification of
lactic acid and ethanol with lipase in the extraction solvent
consisting of Alamine 336 and 2-octanol.
[0017] FIG. 6 is the time course of ethyl lactate synthesis in a
recycle batch packed-bed immobilized lipase reactor under the
following reaction conditions: 10.3 g immobilized lipase from
Candida sp. 99-125, 2.5 ml of 85% (w/w) lactic acid (30 g/l), 8.0
ml of ethanol, 7.5 ml of Alamine 336 and 32 ml of 2-octanol with a
total volume of 50 ml at 25.degree. C. in the bioreactor.
[0018] FIG. 7 shows the long-term operational stability of the
immobilized lipase in a fibrous bed bioreactor at the repeated
batch mode with the reaction conditions: 10.3 g immobilized lipase
from Candida sp. 99-125, 2.5 ml of 85% (w/w) lactic acid (30 g/l),
8.0 ml of ethanol, 7.5 ml of Alamine 336 and 32 ml of 2-octanol
with a total volume of 50 ml at 25.degree. C. in the bioreactor for
24 h for each batch.
[0019] FIG. 8 shows the kinetics of lactic acid ester synthesis
with lactic acid and various alcohols under the reaction
conditions: 0.9 g immobilized Candida sp. 99-125 lipase, 0.5 ml of
85% (w/w) lactic acid (30 g/l), 2.8 M alcohol, 1.5 ml of Alamine
336 and 2-octanol with a total volume of 10 ml at 30.degree. C. and
150 rpm.
[0020] FIG. 9 shows the kinetics of ethyl ester synthesis with
ethanol and various organic acids under the reaction conditions:
0.9 g immobilized Candida sp. 99-125 lipase, 0.33 M acid, 1.6 ml of
ethanol, 1.5 ml of Alamine 336 and 2-octanol with a total volume of
10 ml at 30.degree. C. and 150 rpm.
[0021] FIG. 10 shows two graphs illustrating the kinetics of the
biosynthesis of ethyl butyrate with ethanol and butyric acid with
the butyric acid at various concentrations under the following
reaction conditions: 0.9 g immobilized Candida sp. 99-125 lipase,
2.3 ml of ethanol, 1.5 ml of Alamine 336 and 2-octanol with a total
volume of 10 ml at 30.degree. C. and 150 rpm.
DETAILED DESCRIPTION
[0022] The present invention comprises methods and processes for
producing esters. The methods and processes comprise converting a
fermentable carbon source to organic acids by fermentation with
organic acid producing microorganisms, followed by catalytic
esterification. The methods and processes of the present invention
comprise integrated fermentation, extraction, and esterification
reactions wherein the organic acids produced during fermentation
are extracted into an extraction solvent and then directly reacted
with an alcohol in the presence of a catalyst to form organic
esters. The present invention also relates to methods and processes
of producing esters wherein the organic acids produced during
fermentation and extracted into the extraction solvent are stripped
from the extraction solvent prior to being reacted with an alcohol
in the presence of a catalyst to form organic esters.
[0023] In one embodiment of the present invention, as depicted in
FIG. 1, the fermentation, extraction, and esterification reactions
are integrated. In accordance with this particular embodiment of
the present invention, the fermentation process is carried out by
converting a fermentable carbon source to organic acids by
fermentation with microorganisms, wherein the fermentable carbon
source is derived from biomass feedstocks. Suitable sources of
fermentable carbon sources include any sources of carbon that may
be used in the fermentation process to produce organic acids. In
one specific embodiment, the fermentable carbon source may include
but is not limited to sugars, starch, cellulose and glycerol. In
another specific embodiment, the carbon source may comprise a
carbohydrate source. In yet still a further embodiment, the carbon
source may be derived from biomass feedstocks. Suitable sources of
biomass feedstocks include agricultural residues such as corn
stovers, corn cobs, and rice straw, and processing wastes such as
cheese whey and corn fiber. After sterilization by microfiltration,
cheese whey, which contains mainly lactose, can be hydrolyzed with
lactase immobilized in a fibrous bed reactor, facilitating organic
acid fermentation by organic acid producing microorganisms.
Suitable microorganisms include but are not limited to bacteria,
yeasts, and filamentous fungi. The main byproducts from the corn
milling process are corn fibers and steep liquor, which must be
properly converted to marketable products in order to avoid the
high waste treatment costs due to the high biological oxygen demand
(BOD) content. In addition to sugars (starch, glucose, fructose,
etc.) present in these processing wastes, there are also abundant
sugars (glucose and xylose) present in cellulose and hemicellulose
found in corn stovers, corn cobs, rice straw and many other
agricultural residues and plant biomass. Corn fiber can be
hydrolyzed with dilute HCl to yield glucose and xylose, both of
which can be readily fermented by organic acid producing
microorganisms.
[0024] In one embodiment of the present invention, the fermentation
process is carried out by feeding a fermentable carbon source
derived from feedstock into a bioreactor, such as a fibrous bed
bioreactor as disclosed in U.S. Pat. No. 5,563,069. Conversion of
the fermentable carbohydrates to organic acids is accomplished via
fermentation by organic-acid producing microorganisms.
Additionally, in one embodiment of the present invention, the
fermentation process is conducted at a pH from approximately 4 to
7.
[0025] In one specific embodiment, butyric acid fermentation by
butyric acid producing bacteria is carried out in a fibrous bed
bioreactor. Several species of bacteria can produce butyric acid as
the major fermentation product from a wide range of substrates.
Among them, Clostridium tyrobutyricum possesses several advantages
over other species of bacteria, including high product purity, high
product yield, and simple medium for cell growth. However, the
fermentation reaction may also utilize other butyric acid producing
microorganisms such as: Clostridium butyricum, Clostridium
beijerinckii, Clostridium populeti, and Clostridium
thermobutyricum, as the specific recitation of Clostridium
tyrobutyricum is not meant to limit the scope of the invention.
[0026] However, like other acidogenic bacteria, butyric acid
bacteria are strongly inhibited by their acid products. Thus, in
response to these difficulties, a butyric acid fermentation process
has been developed wherein engineered mutants of Clostridium
tyrobutyricum ATCC 25755 are obtained from inactivating the
chromosomal ack gene encoding acetate kinase, and adapting into a
fibrous bed bioreactor. The Clostridium tyrobutyricum ATCC 25755
are preferably used in fermentation reactions to produce butyric
acid. The Clostridium tyrobutyricum mutants with the inactive
chromosomal ack gene show high butyric acid yield of up to 48%
(w/w), final butyric acid concentration of up to 80 g/L, and high
productivity (>2 g/Lh) of butyric acid from glucose.
[0027] In an alternative embodiment of the present invention,
propionic acid fermentation has also been developed for propionic
acid using the propionic acid producing bacteria Propionibacterium
acidipropionici immobilized in a fibrous bed bioreactor. In this
fermentation, the fermentation pH is maintained at .about.6.0, and
the final propionate concentration reached .about.100 g/L, which is
.about.2.5 times higher than that produced in a conventional
propionic acid fermentation. However, the fermentation reaction may
also utilize other propionic acid producing microorganisms, as the
specific recitation of Propionibacterium acidipropionici is not
meant to limit the scope of the invention.
[0028] In an alternative embodiment of the present invention,
lactic acid fermentation has also been developed for lactic acid
from glucose using the lactic acid producing filamentous fungus
Rhizopus oryzae. The fermentation reaction may be carried out in a
fed-batch extractive fermentation wherein the cells may be
immobilized in a rotating fibrous bed bioreactor. The fermentation
reaction is carried out wherein the pH is maintained at
approximately pH 5. However, the fermentation reaction may also
utilize other lactic acid producing microorganisms, as the specific
recitation of Rhizopus oryzae is not meant to limit the scope of
the invention.
[0029] In addition to the embodiments previously discussed, one
skilled in the art will recognize that similar fermentation
processes can be used to produce various carboxylic acids from
different substrates using different microorganisms, including
bacteria, yeasts, and filamentous fungi.
[0030] Following the fermentation process wherein fermentable
carbohydrates are converted to organic acids by organic acid
producing microorganisms, the organic acids are recovered from the
fermentation broth and purified by extraction using an organic
solvent, i.e. an extractant. In one specific embodiment of the
present invention, the fermentation is preferably coupled with the
extraction. This process, referred to as extractive-fermentation,
allows for continuous production and recovery of the organic acids
produced from the fermentation process in one continuous step.
Extractive-fermentation significantly improves reactor productivity
and final product concentration by reducing end-product inhibition,
thus reducing downstream processing load and recovery costs.
[0031] In one embodiment, extraction of the organic acids in the
fermentation broth may be preferably carried out with an extraction
column. In one specific embodiment, the extraction column may be a
packed extraction column wherein Alamine 336 is the extractant. The
extraction column may alternatively comprise a Karr column. In an
alternative embodiment, extraction of the organic acids in the
fermentation broth may be carried out with a hollow-fiber membrane
extractor.
[0032] In accordance with one embodiment of the present invention,
the extractant is an amine solvent, and is preferably a water
immiscible long-chain aliphatic amine solvent such as Alamine 336.
Among the long-chain aliphatic amines, secondary (e.g., ditridecyl
amine or Adogen 283) and tertiary amines (e.g., tricaprylyl amine
or Alamine 336) are widely used because of their low solubility in
water and high distribution coefficients for carboxylic acids.
However, the organic acids produced during the fermentation process
may also be extracted with other suitable extractants, as the
specific recitation of the previously mentioned aliphatic amines is
not meant to limit the scope of the invention.
[0033] Suitable extractants include those which are biocompatible,
possess high extraction coefficients or K.sub.eq values for the
product, are operable at a pH value close to optimal pH for
fermentation, (usually .about.5 or higher), or possess high
distribution coefficients (K.sub.d). Developing biocompatible
extractants is difficult because solvents with high K.sub.eq values
are usually toxic to cells. Additionally, suitable extractants
should avoid phase separation problems. However, issues concerning
phase separation can be overcome by using a membrane extractor to
prevent direct contact between the extractant and the aqueous
solution.
[0034] With regard to pH, efficient extraction requires an
extractant with pH value below the pK.sub.a value of the organic
acid. Most carboxylic acid fermentations have an optimal pH between
5 and 7. In an extractive-fermentation, there is no requirement
that the pH of the fermentation broth be controlled with the
addition of a base; rather, the pH of the fermentation broth can be
kept at a pseudo-steady-state pH wherein the rate of organic acid
production from the fermentation process is equal to the rate of
organic acid removal by the extraction process. Thus, the removal
of organic acid products by extraction reduces process wastes and
production costs.
[0035] In one specific embodiment of the present invention, an
extractive-fermentation has been developed for butyric acid
production by butyric acid producing bacteria immobilized in a
fibrous bed bioreactor. The butyric acid present in the
fermentation broth can be recovered and purified by extraction
using an aliphatic amine. By coupling the fermentation process with
the extraction process, the resulting extractive-fermentation
process can produce a higher butyrate concentration of >300 g/L
at a higher productivity and purity than the non-coupled
processes.
[0036] In an alternative embodiment of the present invention, an
extractive-fermentation has also been developed for propionic acid
production by Propionibacterium acidipropionici immobilized in a
fibrous bed bioreactor. In this specific embodiment, the
fermentation pH is maintained at .about.4.8, and the final
propionate concentration may reach .about.170 g/L, which is 2.4
times higher than that which may be produced in a comparable
fermentation reaction at pH 7.0.
[0037] As depicted in Table 1 set forth below, this specific
extractive-fermentation may result in not only significantly higher
reactor productivity, but also higher propionate yield and higher
product purity than that of a batch fermentation. These effects may
be attributed to a reduction in the production of acetate and
succinate in the propionic acid fermentation. The increased product
purity may also be attributed to the higher selectivity of amine
extraction for propionic acid than for acetic and succinic
acid.
TABLE-US-00001 TABLE 1 Comparisons of propionic acid production in
extractive and conventional fermentations. Extractive Batch
Fermentation Fermentation pH 7.1 pH 5.0 pH 7.0* pH 5.3 pH 4.8*
Productivity (g/L h) 0.2 0.12 0.09/0.26* 0.98 0.4/2.5* Product
Yield (g/g) Propionic acid 0.31 0.54 0.4-0.65 0.66 0.78 Acetic acid
0.12 0.13 0.10 0.07 0.11 Succinic acid 0.10 0.09 0.09 0.02 0.01 P/A
Ratio 2.58 4.15 4.0 9.8 7.1 Product Purity 58% 71% 69% 88% 88%
Final Propionate 12.5 18.5 71.7 75 170 Concentration (g/L)
*Fermentation with cells immobilized in a fibrous bed bioreactor
(FBB). The higher productivity value is based on the FBB volume,
whereas the lower value is based on the total liquid volume in the
system.
[0038] In yet another embodiment of the present invention, an
extractive-fermentation has been developed for lactic acid from
glucose using the lactic acid producing microorganism Rhizopus
oryzae. The extraction is carried out with Alamine 336 (30% in
oleyl alcohol) followed by back extraction with 6 N NaOH in
hollow-fiber membrane extractors. Lactic acid may be produced
continuously at a stable rate, reaching a concentration of
.about.293 g/L in the stripping solution. The overall lactic acid
yield is higher than 90% based on glucose consumption, with almost
no byproduct produced in the fermentation process. Additionally,
increasing the extractor capacity of the hollow fiber units, which
is proportional to the total membrane surface area, may allow
operation of the fermentation process at a higher pH, resulting in
an increase in reactor productivity.
[0039] In yet still a further embodiment of the present invention,
the organic acids present in the extractant may be separated from
the extractant by stripping. The extractant can then be recycled
back for use in the extraction process. The organic acids present
in the extractant may be stripped by various reagents, including
but not limited to: a base solution (e.g. NaOH), a strong acid
solution (e.g. HCl), hot water, or steam. Stripping is most
preferably accomplished with the use of a base because the base is
energy efficient.
[0040] In accordance with one embodiment of the present invention,
the organic acid extracted from the fermentation broth may be
esterified following the extractive-fermentation. Esterification
comprises reacting an organic acid with an alcohol in the presence
of a catalyst. The catalyst used in the esterification reaction may
include but is not limited to: sulfuric acid, a cation exchange
resin (e.g. Amberlyst 15), or a biocatalyst. In one specific
embodiment of the present invention, the catalyst is preferably an
enzyme, and most preferably a lipase.
[0041] In one embodiment of the present invention, the organic acid
in the extractant may be reacted directly with an alcohol in the
presence of an enzyme, preferably a lipase, to form an ester that
can be readily stripped with steam in a distillation column. The
extractant may be regenerated and recycled for the extraction
process, while the ester and unreacted alcohol may be separated in
the distillation process. To carry out the esterification reaction
in the extractant, an esterification process involving an
immobilized lipase has been developed.
[0042] In one embodiment of the present invention, the
esterification catalyst is preferably an enzyme, and most
preferably a lipase. For esterification, various commercial lipases
such as Novozyme 435 and non-commercial lipases produced either
homologously or heterologously in microorganisms may be used.
Extracellular lipases from Candida lipolytica may also be used for
the esterification process. Among the three extracellular lipases
found in Candida lipolytica, Lipase 2 is responsible for the major
extracellular activity and has been widely used in hydrolysis,
esterification and trans-esterification reactions. Mutants of
Candida lipolytica sp. 99-125 can produce lipase at a high
expression level of 6000 U/mL (1.1 g lipase/L) and with high
productivity of 60 U/h/mL (11 mg/h/L). Mutants of Candida
lipolytica sp. 99-125 can be obtained through a series of classic
mutagenesis reactions.
[0043] In one embodiment of the present invention, the
esterification reaction is carried out by immobilizing an enzyme on
a support surface. More specifically, the esterification reaction
is carried out by immobilizing the enzyme in a fibrous bed
bioreactor. Immobilization of the enzyme involves the following
steps: adsorption of the binding agent to a support surface,
introduction of the enzyme to form aggregates with the binding
agent, and cross linking the enzyme-binding agent aggregates coated
on the support surface.
[0044] The immobilized enzyme reactor has a high productivity and
good long-term stability for the esterification reaction to produce
esters from acids and alcohols. In one specific embodiment of the
present invention, a lipase is preferably immobilized on a support
surface. In accordance with a further embodiment, the lipase is
immobilized on a support surface comprising a fibrous matrix in a
fibrous bed bioreactor. The esterification process with a lipase
immobilized on a support surface can be operated continuously with
a steady product stream for an extended period of months or longer
without significant loss in its productivity. The support surface
may comprise fibrous materials including synthetic fibers, such as
polyester, glass fibers, and natural fibers, such as cotton and
silk. In one specific embodiment wherein the lipase is immobilized
on a support surface, the support surface is preferably a fibrous
material comprising cotton.
[0045] In accordance with this specific embodiment, the binding
agent may comprise but is not limited to alginate and charged
polymers. In a preferred embodiment, the highly branched cationic
polymer polyethyleneimine (PEI) is used. As depicted in FIG. 2, by
preferably binding the enzyme to PEI as a binding agent, the enzyme
may retain almost all of its activity (>90%) with an improved
thermal stability (10 to 20-fold increase). Furthermore, as
depicted in FIG. 2, immobilization of lipase on the fibrous support
matrix is stable even at 60.degree. C. as well as in the organic
media used for the esterification reaction. Additionally, as
depicted in FIG. 2, the immobilized enzyme was stable and retained
almost all of its activity while the free enzyme lost more than 50%
of its activity in 30 minutes.
[0046] In accordance with this specific embodiment, the
cross-linking agent may comprise but is not limited to
glutaraldehyde (GA). Once the enzyme is cross-linked with GA, the
immobilized enzyme is stable and does not leach out from the
support matrix. As previously discussed, FIG. 2 depicts the
construction of an immobilized lipase reactor with an enzyme
immobilized on the fibers.
[0047] In one specific embodiment of the present invention, the
esterification of butyric acid and an alcohol, preferably ethanol
or butanol, can be catalyzed by sulfuric acid, a cation exchange
resin (e.g. Amberlyst 15), or a lipase enzyme. The esterification
reaction is most preferably carried out wherein the catalyst is a
lipase enzyme. Esterification with a lipase may be carried out
under mild reaction conditions, i.e. ambient temperature and
pressure.
[0048] Additionally, esterification with a lipase does not result
in the production of byproducts, with the exception of water. With
proper control on the water content in the reaction medium, a high
product yield of greater than 90% with close to 100% conversion may
be obtained. In one specific embodiment, wherein an ethyl butyrate
ester is produced, a solvent other than ethanol, e.g. n-hexane is
required for lipase catalysis.
[0049] According to embodiments of the present invention, enzymatic
esterification of butyric acid by immobilized lipase in amine
solvent and solvent free systems is feasible. In one specific
embodiment, the organic acid present in the low molecular-weight
tertiary amine, e.g., trialkyl amine, from the extractive
fermentation process can be directly reacted with an alcohol to
produce an ester. As depicted in FIG. 3, in one particular
embodiment, about 65% conversion of butyric acid present in butanol
to its ester may be achieved by immobilized lipase in a fibrous bed
bioreactor wherein the butyric acid is not stripped from the
extractant prior to esterification. In an alternative embodiment of
the present invention, esterification of butyric acid in a solvent
free system may occur when butanol is the alcohol substrate to
produce butyl butyrate.
[0050] In accordance with one embodiment, as depicted in FIG. 3, a
high ethyl butyrate concentration of 1.2 M (140 g/L) and butyl
butyrate of 1.3 M (180 g/L) may be obtained with a conversion of
60% after 24 hours reaction in the amine solvent. In an alternative
embodiment, a high butyl butyrate concentration of 1.9 M (272 g/L)
may be obtained with a high conversion of 87% after 24 hours
reaction in a solvent free system.
[0051] In one embodiment of the present invention wherein the
esterification reaction takes place in the presence of an amine
solvent, the reaction products, i.e. the ester and unreacted
alcohol, are stripped from the extractant. More specifically, the
ester and unreacted alcohol are stripped from the amine solvent
with steam and are then separated in a distillation column. The
amine solvent is thus regenerated and recycled for the extraction
process as shown in FIG. 1.
[0052] In accordance with one specific embodiment of the present
invention, as depicted in FIG. 4, colorless ethyl butyrate and
butyl butyrate esters may be obtained from the reaction mixture by
using distillation to separate the ester from the amine solvent and
unreacted acid and alcohol.
Example 1
Materials and Methods
[0053] Enzymes and Reagents. The lipase from Candida sp. 99-125 was
produced in a fermentation process and then immobilized on cotton
cloth, which was dried at room temperature and stored at 4.degree.
C. until use. The organic solvent consisted of Alamine 336
(straight chain tertiary amine containing C.sub.8-C.sub.10 alkyl
groups, Henkel Corp. USA) and 2-octanol as the diluent. Unless
otherwise noted, all chemicals, including lactic acid (85% w/w),
ethyl lactate, isopropyl lactate and butyl lactate, used in this
work were of analytical grade (Sigma, St. Louis, Mo.).
[0054] Esterification in Shake-Flasks. Ester synthesis was carried
out in 100 ml stoppered flasks with 10 ml of reaction mixture. The
reaction was performed with 0.33 M lactic acid, 2.8 M ethanol, 0.33
M Alamine 336, 7 ml 2-octanol and 0.9 g immobilized lipase from
Candida sp. 99-125, with a total volume of 10 ml. The mixture was
incubated for 48 h in an orbital shaker at 30.degree. C. and 150
rpm. Samples were taken at regular intervals and ethyl lactate and
lactic acid were measured by using HPLC. All experiments were
carried out in duplicate and mean values were reported.
[0055] Production of Ethyl Lactate in a Fibrous-Bed Bioreactor. The
lipase from Candida sp. 99-125 was produced in a fermentation
process and was then immobilized on cotton cloth. Lipase was
produced at a high expression level of 6000 U/mL (1.1 g lipase/L)
and high productivity of 60 U/h/mL (11 mg/h/L). The cotton cloth
was dried at room temperature and stored at 4.degree. C. until use.
Approximately 10.3 g of cotton cloth with immobilized lipase from
Candida sp. 99-125 were packed in a column.
[0056] A reaction solution consisting of 0.33 M lactic acid, 2.8 M
ethanol, 0.33 M Alamine 336 (straight chain tertiary amine
containing C.sub.8-C.sub.10 alkyl groups, Henkel Corp. USA), and 35
ml 2-octanol, with a total volume of 50 ml, in an Erlenmeyer flask
was recirculated through the packed column at 25.degree. C. The
flow rate was set at 5 mL/min. Samples were taken at regular
intervals and the concentrations of ethyl lactate and lactic acid
were analyzed by using HPLC. For long-term study to evaluate the
operational stability, the reaction solution in the system was
replaced with a fresh reaction solution every 24 h for 8 days. The
reactor was then left idling in the room temperature until it was
run again with a new batch of fresh reaction solution on day
21.
[0057] As depicted in FIG. 8, a study of the kinetics of lactic
acid ester synthesis with lactic acid and various alcohols under
the following reaction conditions was conducted: 0.9 g immobilized
Candida sp. 99-125 lipase, 0.5 ml of 85% (w/w) lactic acid (30
g/l), 2.8 M alcohol, 1.5 ml of Alamine 336 and 2-octanol with a
total volume of 10 ml at 30.degree. C. and 150 rpm.
Example 2
Butyric Acid Production
[0058] An extractive-fermentation for butyric acid production from
glucose by immobilized cells of Clostridium tyrobutyricum in a
fibrous bed bioreactor was conducted. The extractant consisted of
10% (v/v) Alamine 336 in oleyl alcohol. The process was contained
within a hollow-fiber membrane extractor to selectively remove
butyric acid from the fermentation broth. The extractant was
simultaneously regenerated by stripping with NaOH in a second
membrane extractor. The fermentation pH was self-regulated by a
balance between butyric acid production and removal of butyric acid
by extraction, and was kept at .about.pH 5.5 under the conditions
studied. Compared to the conventional fermentation, the extractive
fermentation gave a higher product concentration of >300 g/L and
product purity of 91%. Extractive-fermentation also gave a higher
reactor productivity of 7.37 g/Lh and butyric acid yield of 0.45
g/g.
[0059] For comparison, the same fermentation without on-line
extraction to remove butyric acid resulted in a final butyric acid
concentration of .about.43.4 g/L, a butyric acid yield of 0.42 g/g,
and a reactor productivity of 6.77 g/Lh when the pH was 6.0. When
the pH was 5.5, the final butyric acid concentration was 20.4 g/L,
the butyric acid yield was 0.38 g/g, and the reactor productivity
was 5.11 g/Lh. The improved performance for the extractive
fermentation can be attributed to reduced product inhibition by
selectively removing butyric acid from the fermentation broth. The
solvent was found to be toxic to free cells in suspension, but not
harmful to cells immobilized in the fibrous bed bioreactor. The
process was stable and gave consistent long-term performance for
the entire 2-week period studied.
[0060] The butyric acid present in the extractant may be stripped
by various methods, including stripping with a base solution (e.g.
NaOH), a strong acid solution (e.g. HCl), or with hot water or
steam. The butyric acid in the solvent also can be reacted directly
with an alcohol to form an ester under the catalytic action of a
lipase.
Example 3
Esterification
[0061] An integrated fermentation, extraction and esterification
process, as depicted in FIG. 1, was employed to produce esters from
alcohols and organic acids produced in fermentation. Butyric acid
was first extracted into an amine solvent and was then reacted with
butanol to form butyl butyrate ester. In this process, the
stripping step was replaced with esterification, employing an
alcohol and catalyst to catalyze the reaction between alcohol and
organic acids present in the extractant. More specifically, in this
process, the organic acids present in the extractant were directly
reacted with alcohol to produce ester. As depicted in FIG. 3, more
than 60% conversion of butyric acid to its ester with ethanol or
butanol was achieved with the reaction in an organic solvent. The
ester present in the amine solvent was separated by distillation or
other methods and the amine solvent was then recycled back for use
in the extraction process, as depicted in FIG. 1. A solvent free
system was also employed when butanol is the alcohol substrate to
produce butyl butyrate with a conversion of .about.90%.
[0062] In the case of ethyl butyrate production, solvents other
than ethanol (e.g. n-hexane) were needed for lipase catalysis.
Esterification of butyric acid with butanol present in an organic
solvent such as Alamine 336 was accomplished via the use of a
lipase, preferably immobilized on a solid support.
[0063] As compared to free lipase, immobilized lipase offered many
benefits, including enzyme reuse, easy separation of product from
enzyme and the potential to run continuous processes via packed-bed
reactors. Immobilized lipase had a shift toward a higher optimal
temperature than that of free lipase. Also, the immobilized lipase
esterification process was able to be operated continuously with a
very steady product stream for an extended period of months or
longer without significant loss in its productivity. FIG. 3 depicts
the kinetics of esterification of butyric acid with ethanol (top)
or butanol (bottom) by immobilized lipase in a fibrous bed
bioreactor. As depicted in FIG. 3, a high ethyl butyrate
concentration of 1.2 M (140 g/L) and butyl butyrate of 1.3 M (180
g/L) were obtained with a conversion of 60% after 24 h reaction in
the amine solvent. Also, a high butyl butyrate concentration of 1.9
M (272 g/L) was obtained with a high conversion of 87% in a
solvent-free system.
[0064] As depicted in FIG. 2, an immobilized lipase reactor was
constructed with lipase immobilized on the fibers. The immobilized
enzyme was stable at 60.degree. C. and retained almost all of its
activity while the free enzyme lost more than 50% of its activity
in 30 minutes.
[0065] Immobilized lipase from Candida sp. 99-125 showed good
catalytic ability for esterification of lactic acid. In general,
increasing enzyme loading resulted in an increase in ester yield.
The conversion rate for lactic acid to ethyl lactate ester
increased from 18.5% at 0.45 g of lipase to 24% at 0.9 g lipase.
Ethyl lactate was the only ester detected. Ethyl lactate was
continuously produced in a plug-flow reactor for 21 days without
significant decrease in the outlet product concentration,
suggesting that the extractant was not toxic to lipase used in the
esterification reaction. The results indicated that enzymatic
esterification could be successfully carried out in an organic
solvent.
[0066] FIG. 5 depicts the reaction mechanism of the esterification
of lactic acid and ethanol with lipase in the extraction solvent
consisting of Alamine 336 and 2-octanol. Novozyme 435 and
immobilized lipase from Candida sp. 99-125 were used as catalysts
for the esterification of lactic acid and ethanol. The preference
of Novozyme 435 for the different enantiomers of lactic acid was
previously investigated, finding that Novozyme 435 gives equal rate
toward both enantiomers of lactic acid. Lipase from Candida sp.
99-125 was also found to catalyze the esterification of both
enantiomers of lactic acid at almost equal rates. However, in the
extraction solvent, lactic acid was found to exist as both an
Alamine 336-lactic acid complex and as free lactic acid. Only free
lactic acid was found to react with ethanol to produce ethyl
lactate.
[0067] 2-octanol was also found to react with lactic acid to form
2-octyl lactate. Esterification between 2-octanol and lactic acid
was analyzed under various reaction systems. The conversion of
2-octanol to 2-octyl lactate was found to be 10.5% in 2-octanol
without ethanol, whereas no 2-octyl lactate was detected in the
reaction when 4M ethanol was present, suggesting that the lipase
from Candida sp. 99-125 has a much lower activity towards secondary
alcohol than primary alcohol.
Example 4
Production of Organic Acid Esters by Lipase in Extractant
[0068] The reaction kinetics of enzymatic esterification were
studied with immobilized lipase from Candida sp. 99-125 in an
extraction solvent used in extracting carboxylic acids from the
fermentation broth. The effects of solvent concentration, molecular
sieve for water removal, acid concentration, and molar ratio of
alcohol to acid on the conversion of lactic acid to ethyl lactate
were investigated. Ethyl lactate was continuously produced in a
plug-flow reactor for 21 days without significant decrease in the
outlet product concentration, suggesting that the extractant was
not toxic to lipase used in the esterification reaction. The
results indicated that enzymatic esterification could be
successfully carried out in an organic solvent to produce organic
acid esters from a fermentable carbon source and alcohols.
[0069] Effect of the Extractant. Various organic solvents were
investigated for their effects on the synthesis of ethyl lactate
and the results are shown in Table 2 set forth below. The highest
conversion (74%) was obtained in acetone, followed by the
conversion of 63% and 33% in 2-octanol and 0.33 M Alamine 336 in
2-octanol, respectively. Cyclohexane and hexane with high log P
value gave very low conversion. Cyclohexane and hexane, like other
apolar solvents, were unable to completely dissolve lactic acid.
Low lactic acid concentration in the solvent may have resulted in a
low conversion. Undissolved lactic acid can deactivate lipase
because of the high acidity of lactic acid. Another reason for the
low conversion is that high water content [15% (w/w)] in lactic
acid solution could produce saturation of high hydrophobic
solvents, resulting in the shift of reaction equilibrium towards
hydrolysis. High solubility of lactic acid in acetone and 2-octanol
might contribute to the higher conversion to ester.
[0070] Lower conversion and initial rate were observed when Alamine
336 was added into 2-octanol. Fifty-one percent and 35% conversion
was obtained in 0.15 M and 0.33 M Alamine, respectively, and 0.66 M
Alamine 336 achieved only 6% conversion. Improvements in the
initial rate and conversion were reported with the addition of
trioctylamine into the hydrolysis reaction system of dynamic
kinetic resolution of (R,S)-profen 2,2,2-trifluoroethyl thioesters
using Candida rugosa lipase. The improvement was attributed to the
ion-pair formation between the organo-soluble base and the product
acid, which could prevent the acid inhibition and shift the
reaction towards the products. Alamine 336 was found to react with
lactic acid to form the ion-pair complex which resulted in a shift
of the reaction towards the left-hand side (hydrolysis side),
resulting in a lower ester conversion (see FIG. 5). Without using
an organic solvent, the esterification reaction exhibited a low
conversion of 5%, possibly due to the inactivation of lipase by
lactic acid (acid inactivation) or excessive ethanol
(dehydration).
TABLE-US-00002 TABLE 2 Effects of organic solvents on the
production of ethyl lactate. Reaction rate Conversion Solvent Log P
(.mu.mol/h) (%) No solvent.sup.a -- 1.2 5 Acetonitrile -0.33 10.9
16 Acetone -0.24 92.7 74 n-Hexane 3.5 2.7 4 Cylcohexane 3.2 3.4 8
2-octanol 2.8 227.7 63 7.5% (v/v) Alamine 336 in 2-octanol -- 229.5
51 15% (v/v) Alamine 336 in 2-octanol -- 136.8 35 30% (v/v) Alamine
336 in 2-octanol -- 10.4 6 Reaction conditions: 0.9 g immobilized
Candida sp. 99-125 lipase, 0.5 ml of 85% (w/w) lactic acid (30
g/l), 1.6 ml of ethanol, 1.5 ml of Alamine 336 and 6.4 ml of
organic solvents with a total volume of 10 ml at 30.degree. C. and
150 rpm for 48 h. .sup.aNo solvent: ethanol was added instead of
organic solvents.
[0071] Effect of Ethanol Concentration. The effect of molar ratio
of alcohol to acid on conversion of lactic acid to ester was
studied. The conversion increased from 20% to 44% when the molar
ratio of alcohol to acid increased from 0.5 to 16. Two reasons may
explain the effect of molar ratio on conversion. First, excessive
ethanol can drive the esterification reaction towards the products
and result in a higher conversion. At the same time, excessive
ethanol can also change solvent characteristics such as log P that
can affect the activity of lipase. Excessive ethanol can also
dissolve the water initially present in lactic acid (15%) and water
produced during esterification, which can also drive the reaction
towards ester.
[0072] Effect of Water Absorbent. Because hydrolysis is the reverse
of esterification, the degree of hydration of solvent plays an
important role in esterification conversion. Thus, the effect of
water absorbent on the conversion was studied. Molecular sieve was
added into the reaction system to remove water formed during
esterification to drive the reaction towards products. Adding 1.0 g
of molecular sieve to the reaction solution increased the
conversion by about 5%.
[0073] Effects of Lactic Acid and Alamine 336 Concentrations. As
set forth below, Table 3 shows the effects of lactic acid and
Alamine 336 concentrations on the initial rate and conversion of
ethyl lactate. Conversion and initial rate decreased with
increasing the Alamine 336 concentration when lactic acid
concentration was low (0.15 M and 0.33 M). Low conversion and
initial rate were obtained at different concentrations of lactic
acid from 0.15 M to 1 M when a high Alamine 336 concentration of
0.66 M was used. A high lactic acid concentration of 1 M also gave
a very low initial rate and conversion at different Alamine 336
concentrations. The optimum lactic acid concentrations at different
Alamine 336 concentrations of 0.15 M, 0.33 M and 0.66 M was 0.33 M,
0.5 M and 0.5 M, respectively.
[0074] Three reasons may explain the effect of Alamine 336
concentrations. First, organic base of Alamine 336 can react with
lactic acid to form the ion-pair complex, which reduces the amount
of free lactic acid (reactive lactic acid) resulting in lower
initial rate and conversion. Second, the addition of Alamine 336
can suppress the high acidity of lactic acid which deactivates the
lipase activity. Finally, high concentrations of Alamine and lactic
acid can change the characteristics of solvent, thus changing the
solvation of the reaction components.
TABLE-US-00003 TABLE 3 Effects of lactic acid and Alamine 336
concentrations on ester conversion. Lactic acid 7.5% (v/v) Alamine
336 15% (v/v) Alamine 336 30% (v/v) Alamine 336 concentration
Initial rate Conversion Initial rate Conversion Initial rate
Conversion (g/L) (.mu.mol/h/g) (%) (.mu.mol/h/g) (%) (.mu.mol/h/g)
(%) 15 53.8 30.6 19.4 10.2 1.0 4.9 30 180 54 135 30.5 24.9 11.8 45
189 31 215 37.1 113 20.2 60 44.4 4.8 173 25.9 88.3 13.5 90 0.9 1.9
57.7 4.8 25.0 3 Reaction conditions: 0.9 g immobilized Candida sp.
99-125 lipase, 1:8 molar ratio of lactic acid to ethanol, 2-octanol
with a total volume of 10 ml at 30.degree. C. and 150 rpm.
[0075] Long-Term Production of Ethyl Lactate in a Fibrous Bed
Bioreactor. FIG. 6 shows the kinetics of esterification with
immobilized lipase in a fibrous bed bioreactor under recycle batch
conditions. The ethyl lactate conversion was 39% after 44 h. The
operational stability of the enzyme was investigated for 21 days by
operating the reactor in the repeated batch mode. As depicted in
FIG. 7, the immobilized lipase was stable and there was no
significant loss in enzyme activity as indicated by the stable
conversion rate during the entire period of 21 days studied.
Additionally, as depicted in FIG. 7, the immobilized lipase was
stable and there was no significant loss in enzyme activity as
indicated by the stable conversion rate during the entire period of
21 days studied.
[0076] Effect of Alcohol Chain Length on Esterification. As
depicted in FIG. 8, a study was conducted on the use of alcohols
with different chain lengths to synthesize different lactate
esters. When butanol and 1-octanol were used as the acyl acceptor,
the conversion was 54%, which is higher than the conversion
obtained for ethyl lactate (37%). Secondary alcohols such as
isopropanol gave a very low conversion of 7.7%. This result shows
that Candida sp. 99-125 lipase is more active with primary and
medium-chain alcohols.
[0077] Production of Organic Acid Esters. The technology that
couples extractive fermentation with enzymatic esterification can
also be applied to ethyl butyrate and ethyl propionate production.
FIG. 9 shows the synthesis of ethyl ester with ethanol and various
short chain fatty acids. High conversions of 64% and 70% were
obtained when butyric acid and propionic acid were used as the acyl
donor, respectively. Acetic acid gave the lowest ester conversion
of 22%. The conversion increased with an increase in the carbon
chain length of the acid molecule.
[0078] This indicates that this lipase has a higher activity toward
long-chain fatty acids. Water initially present in lactic acid
solution can shift the reaction toward hydrolysis and resulted in
low ethyl lactate conversion. The effect of butyric acid
concentration on conversion is shown in FIG. 10. The initial rate
and ethyl butyrate concentration increased with increasing the
butyric acid concentration (up to 3 M), indicating that high
butyric acid concentration would not inhibit or deactivate the
enzyme.
[0079] From above examples, it is apparent that an integrated
process with fermentation, extraction, and esterification units can
produce various organic esters from organic acids and alcohols
produced in fermentation. The organic acid is first extracted into
an amine solvent and then reacted with alcohol to form the ester.
In this process, the ester present in the amine solvent can be
readily separated by steam stripping, and the amine solvent can
then be recycled back for use in the extraction process. Further
separation and purification of ester can be done by distillation,
pervaporation, or nanofiltration, with the former being the
preferred choice because it is commonly used in ethanol and butanol
production plants.
[0080] Extractive fermentation to selectively separate the
desirable product, such as butyric acid, in situ has the advantages
of reducing product inhibition and increasing the fermentation rate
and product yield. By selectively removing butyric acid from the
fermentation broth continuously, the fermentation pathway may be
shifted to produce more butyric acid and less of the byproducts
(e.g., acetic acid), which also make product recovery and
purification easier and less costly. Additionally, conducting
enzymatic esterification in the extractant containing the
fermentation produced butyric acid can dramatically reduce
production costs for butyrate esters since there are no costly
separation or purification steps involved in the process. Although
the above examples substantially focus on butyric acid, the same
technology described herein may also be applied to many other
organic acids, i.e., acetic acid, propionic acid, lactic acid,
citric acid, succinic acid, fumaric acid, itaconic acid, and
long-chain fatty acids. The same technology may also be used to
produce flavor ester compounds, such as amyl butyrate, and
biodiesel from fusil oils and long-chain fatty acids present in
food wastes.
[0081] With the fibrous bed bioreactor and extractive fermentation
coupled with enzymatic esterification, high product yield,
concentration, and reactor productivity can be achieved to reduce
the product cost to a competitive level for commercial application,
thus benefiting the bio-based industry by providing a viable avenue
for better byproduct utilization and high-value products suitable
for various markets. Additionally, the present invention may also
economically convert fermentation produced butyric acid and ethanol
to ethyl butyrate ester, which may be used as a biofuel.
[0082] It is noted that terms like "preferably," "generally,"
"commonly," and "typically" are not utilized herein to limit the
scope of the claimed invention or to imply that certain features
are critical, essential, or even important to the structure or
function of the claimed invention. Rather, these terms are merely
intended to highlight alternative or additional features that may
or may not be utilized in a particular embodiment of the present
invention.
[0083] For the purposes of describing and defining the present
invention it is noted that the term "substantially" is utilized
herein to represent the inherent degree of uncertainty that may be
attributed to any quantitative comparison, value, measurement, or
other representation. The term "substantially" is also utilized
herein to represent the degree by which a quantitative
representation may vary from a stated reference without resulting
in a change in the basic function of the subject matter at
issue.
[0084] Having described the invention in detail and by reference to
specific embodiments thereof, it will be apparent that
modifications and variations are possible without departing from
the scope of the invention defined in the appended claims. More
specifically, although some aspects of the present invention are
identified herein as preferred or particularly advantageous, it is
contemplated that the present invention is not necessarily limited
to these preferred aspects of the invention.
* * * * *